Abstract
Studying the metabolism of prohibited substances is an essential element in anti-doping research in order to facilitate and improve detectability. Whilst pharmacokinetic studies on healthy volunteers are valuable, they are often difficult, not least due to safety reasons and ethical constraints, especially concerning peptidic substances, which must be administered parenterally. Hence, there is a growing need for suitable in vitro models and sophisticated analytical strategies to investigate the metabolism of protein- and peptide-derived drugs. These include human growth hormone (hGH) and its main mediator insulin-like growth factor-I (IGF-I), both prohibited in professional sports for their anabolic and lipolytic effects, while challenging in their detection, as they occur naturally in the human body.Within this study, the in vitro metabolism of hGH and IGF-I was investigated using a stable-isotope-labelled reporter ion screening strategy (IRIS). A combination of liquid chromatography, high-resolution mass spectrometry, and characteristic immonium ions generated by internal dissociation of the stable-isotope-labelled peptidic metabolites enabled the detection of specific fragments. Several degradation products for hGH and IGF-I were identified within this study. These metabolites, potentially even indicative for subcutaneous administration of the drugs, could serve as promising targets for the detection of hGH and IGF-I misuse in future anti-doping applications.
Highlights
The continuing progress of modern anti-doping analytics has allowed for great achievements in the last few years, enabling the detection of infinitesimal amounts of prohibited substances and even the establishment of strategies to uncover methods such as gene doping or autologous blood transfusion [1]
The in vitro metabolism of human growth hormone (hGH) and insulin-like growth factor-I (IGF-I) in different biological fluids was investigated by a combination of liquid chromatography–high-resolution mass spectrometry (LC–HRMS) and ion screening strategy (IRIS), using uniformly stable-isotope-labelled peptides to detect specific metabolites and subsequently resolve their structure via MS/MS-experiments
Investigating the metabolism of peptide- and protein-derived drugs presents a challenge in anti-doping research and analytical chemistry
Summary
The continuing progress of modern anti-doping analytics has allowed for great achievements in the last few years, enabling the detection of infinitesimal amounts of prohibited substances and even the establishment of strategies to uncover methods such as gene doping or autologous blood transfusion [1]. In terms of toxicity or bioavailability, this is essential for drug research and development, but it has led to an increasing number of studies examining in vitro protein and peptide metabolism in the context of anti-doping research, with a particular interest in developing new methods of detecting drug abuse [10,11,12,13,14,15,16] Peptides included in these studies were, amongst others, insulin, synacthen, and several growth hormone-releasing peptides with, for example, a representative metabolite of insulin generated in vitro being detected within in vivo samples obtained after subcutaneous administration of the drug [12,13,15]. The in vitro metabolism of hGH and IGF-I in different biological fluids was investigated by a combination of liquid chromatography–high-resolution mass spectrometry (LC–HRMS) and IRIS, using uniformly stable-isotope-labelled peptides to detect specific metabolites and subsequently resolve their structure via MS/MS-experiments.
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